Heterotrimeric (alpha-beta-gamma) Gs mediates agonist-induced stimulation of adenylyl cyclase (AC). The alpha subunit (Gs-alpha) has intrinsic GTPase activity. Gs is activated when Gs-alpha binds GTP or a GTP analog (e.g. GTP-gamma-S, Gpp[NH]p, or Gpp[CH2]p) and it is deactivated when GTP is hydrolyzed. Naturally occurring mutations of Gs-alpha that suppress Gs activity lead to a human disease called pseudohypoparathyroidism (PHP). Patients with PHP do not respond to hormones that use Gs-coupled receptors, particularly parathyroid hormone. The outcome of being refractory to these hormones is that patients with PHP have a collection of maladies labeled as Albright hereditary osteodystrophy which include short stature, obesity, subcutaneous ossifications, and mental retardation. Our investigations indicate that mutations which cause PHP either 1) reduce the affinity of Gs-alpha for GDP which destabilize the protein and causes it to be denatured at physiological temperatures, or 2) reduce the half-life of the activated conformation of Gs so that agonist-mediated stimulation of AC is attenuated. For decades it has been known that Gs activation in solution can be accompanied by dissociation of Gs-alpha from the G protein beta-gamma subunit complex (G-beta-gamma). However, our recent evidence suggests that subunit dissociation is caused by non- physiological conditions rather than activation. Nevertheless, the prevalent view is that subunit dissociation also occurs when Gs is activated in situ despite a lack of evidence. Cholera toxin (CTx) will ADP-ribosylate Gs-alpha. Gs-alpha-deficient cyc- membranes were stripped of G-beta-gamma. When the stripped cyc- membranes were incubated with Gs-alpha and/or G-beta-gamma, each was incorporated into the membranes independently of the other. Both Gs-alpha and G-beta- gamma had to be present the in membranes, and they had to be able to form a heterotrimer in order for CTx to ADP-ribosylate Gs-alpha, indicating that the membrane bound Gs heterotrimer is a substrate for CTx, but the Gs-alpha subunit by itself is not. When Gs-alpha was completely and irreversibly activated with GTP-gamma-S and incorporated into stripped cyc- membranes it was a poor substrate for CTx and a weak stimulator of AC unless G-beta-gamma was also incorporated. Furthermore, the level of AC stimulation corresponded to the amount of Gs heterotrimer that was formed in the membranes from GTP-gamma-S- activated Gs-alpha and G-beta-gamma indicating that AC is stimulated by an activated Gs heterotrimer in cell membranes. Heterotrimeric Gi mediates agonist-induced inhibition of AC. Surface plasmon resonance spectroscopy (SPR) is being used to determine the affinity of Gi subunits for each other. When GDP is bound to Gi-alpha the equilibrium dissociation constant for G-beta-gamma is approximately 10 nM. Gi subunit affinity is decreased by non-hydrolyzable GTP analogs, and varies depending upon the analog that is bound to Gi-alpha. Qualitatively, the affinity of Gi-alpha for G-beta-gamma is less when GTP-gamma-S is bound, than when Gpp[NH]p is bound, which is in turn less than when Gpp[CH2]p is bound. If GTP fits the pattern then its effects on Gi subunit affinity can not be equated with those of its analogs as has been done extensively in the past. - heterotrimeric G proteins, adenylyl cyclase, cholera toxin, surface plasmon resonance